Of the dozen or so solved structures of voltage-gated ion channels (VGICs), only those of a weakly voltage-dependent Arabidopsis channel show the channel in its resting, de-activated state. Structures of VGIC voltage sensors in their resting ?down? position are lacking because when solubilized in detergent or even reconstituted into nanodiscs, the absence of a membrane potential always leaves the voltage sensors in an activated or inactivated-relaxed conformation. In the proposed work innovative single-particle cryo-EM methods will be applied to obtain structures VGIC proteins as reconstituted into lipid vesicles. In the previous funding period we have made many methodological advances and have progressed to the point of obtaining nanometer/sub-nanometer cryo-EM structures of the Kv1.2 wildtype and ?paddle chimera? potassium channel constructs, reconstituted into vesicles. In these structures no membrane potential is applied. We now propose to extend that work to higher resolution and to the resting-state structure of Kv1.2, the best-understood mammalian VGIC, by subjecting it to a large negative membrane potential. We will then acqure resting-state and activated-state structures of the Nav1.7 sodium channel, which is of intense interest as a central player and drug target in pain sensation. These structures will enable for the first time a quantitative understanding of VGIC voltage sensing, and mechanisms of pain disorders due to sodium channel mutations.